Bottom Line:
However, the persistent translation of certain mRNAs is required for deployment of an adequate stress response.Although this led to a 5.4-fold general translational repression, the protein coding open reading frames (ORFs) of certain individual mRNAs exhibited resistance to the inhibition.Phylogenetic analysis suggests that at least two regulatory uORFs (namely, in SLC35A4 and MIEF1) encode functional protein products.

ABSTRACTEukaryotic cells rapidly reduce protein synthesis in response to various stress conditions. This can be achieved by the phosphorylation-mediated inactivation of a key translation initiation factor, eukaryotic initiation factor 2 (eIF2). However, the persistent translation of certain mRNAs is required for deployment of an adequate stress response. We carried out ribosome profiling of cultured human cells under conditions of severe stress induced with sodium arsenite. Although this led to a 5.4-fold general translational repression, the protein coding open reading frames (ORFs) of certain individual mRNAs exhibited resistance to the inhibition. Nearly all resistant transcripts possess at least one efficiently translated upstream open reading frame (uORF) that represses translation of the main coding ORF under normal conditions. Site-specific mutagenesis of two identified stress resistant mRNAs (PPP1R15B and IFRD1) demonstrated that a single uORF is sufficient for eIF2-mediated translation control in both cases. Phylogenetic analysis suggests that at least two regulatory uORFs (namely, in SLC35A4 and MIEF1) encode functional protein products.

fig2s3: Publicly available ribosome profiling data in GWIPS-viz forSLC35A4 and MIEF1.Ribosome profiling data aligned to the SLC35A4(A) and MIEF1 (B) loci ofthe human genome from nine studies available in the GWIPS-viz Browser.The positions of the conserved upstream open reading frames (uORFs) areshown with a red bar below the blue bars representing correspondingRefSeq transcripts.DOI:http://dx.doi.org/10.7554/eLife.03971.011

Mentions:
To our knowledge the other newly identified mRNAs have not been shown to be regulatedat the translational level before. One of the most unusual examples is the mRNAencoding the probable UDP-sugar transporter protein SLC35A4 (Figure 2). Its 5′ leader contains 11 AUG codons, most ofwhich are not conserved; however, one AUG that initiates a 102 codon uORF is highlyconserved across vertebrates. This uORF encodes a peptide sequence containing PFAMdomain DUF4535, ID PF15054; moreover, the pattern of its conservation is consistentwith protein coding evolution (Figure2—figure supplement 1), suggesting that this uORF likely encodes afunctional protein. This alternative protein (EMBL accession HF548106) was recentlydetected by mass spectrometry analysis of cultured cells and human tissues (Vanderperre et al., 2013; Kim et al., 2014). We examined translation of this mRNA inother publicly available ribosome profiling datasets using GWIPS-viz (Michel et al., 2014) and found that this uORFis translated in all datasets (Figure2—figure supplement 3A). How ribosomes reach the 12th AUGcodon upon arsenite treatment is unclear and merits further investigation.

fig2s3: Publicly available ribosome profiling data in GWIPS-viz forSLC35A4 and MIEF1.Ribosome profiling data aligned to the SLC35A4(A) and MIEF1 (B) loci ofthe human genome from nine studies available in the GWIPS-viz Browser.The positions of the conserved upstream open reading frames (uORFs) areshown with a red bar below the blue bars representing correspondingRefSeq transcripts.DOI:http://dx.doi.org/10.7554/eLife.03971.011

Mentions:
To our knowledge the other newly identified mRNAs have not been shown to be regulatedat the translational level before. One of the most unusual examples is the mRNAencoding the probable UDP-sugar transporter protein SLC35A4 (Figure 2). Its 5′ leader contains 11 AUG codons, most ofwhich are not conserved; however, one AUG that initiates a 102 codon uORF is highlyconserved across vertebrates. This uORF encodes a peptide sequence containing PFAMdomain DUF4535, ID PF15054; moreover, the pattern of its conservation is consistentwith protein coding evolution (Figure2—figure supplement 1), suggesting that this uORF likely encodes afunctional protein. This alternative protein (EMBL accession HF548106) was recentlydetected by mass spectrometry analysis of cultured cells and human tissues (Vanderperre et al., 2013; Kim et al., 2014). We examined translation of this mRNA inother publicly available ribosome profiling datasets using GWIPS-viz (Michel et al., 2014) and found that this uORFis translated in all datasets (Figure2—figure supplement 3A). How ribosomes reach the 12th AUGcodon upon arsenite treatment is unclear and merits further investigation.

Bottom Line:
However, the persistent translation of certain mRNAs is required for deployment of an adequate stress response.Although this led to a 5.4-fold general translational repression, the protein coding open reading frames (ORFs) of certain individual mRNAs exhibited resistance to the inhibition.Phylogenetic analysis suggests that at least two regulatory uORFs (namely, in SLC35A4 and MIEF1) encode functional protein products.

ABSTRACTEukaryotic cells rapidly reduce protein synthesis in response to various stress conditions. This can be achieved by the phosphorylation-mediated inactivation of a key translation initiation factor, eukaryotic initiation factor 2 (eIF2). However, the persistent translation of certain mRNAs is required for deployment of an adequate stress response. We carried out ribosome profiling of cultured human cells under conditions of severe stress induced with sodium arsenite. Although this led to a 5.4-fold general translational repression, the protein coding open reading frames (ORFs) of certain individual mRNAs exhibited resistance to the inhibition. Nearly all resistant transcripts possess at least one efficiently translated upstream open reading frame (uORF) that represses translation of the main coding ORF under normal conditions. Site-specific mutagenesis of two identified stress resistant mRNAs (PPP1R15B and IFRD1) demonstrated that a single uORF is sufficient for eIF2-mediated translation control in both cases. Phylogenetic analysis suggests that at least two regulatory uORFs (namely, in SLC35A4 and MIEF1) encode functional protein products.